SE430507B - PROCEDURE FOR PREPARING A 9-HYDROXYETOXIMETHYL GUANINE DERIVATIVE - Google Patents

Description

15 20 25 30 35 7802140-9 2 aluminum / 3 or ammonium. The compounds of formula (I) wherein Z is sodium, potassium or ammonium and W is hydrogen are preferred, and the compounds of formula (I) wherein Z is sodium or ammonium and W is hydrogen are particularly preferred.

In the above definition of W and Z, the polyvalent cations are expressed as calcium / 2, magnesium / 2 and aluminum / 3, which is meant to mean the cation divided by its valence, ie Ca ++ / 2, Mg ++ / 2 and Al +++ / 3. This is intended to indicate that the calcium or magnesium cations are bound by ionic bonding to two phosphate acids, and aluminum to three.

The process for the preparation of a compound of formula (I), as defined above, involves reacting (a) a compound of formula (II) with HZN 1 H2.0.CH2.CH2.0H with a phosphorylating agents to give a compound of formula (I), wherein both W and Z are hydrogen atoms; (b) a compound of formula (III) N / N (II)) is subjected to ammonolysis to give a compound of formula (III). I), and optionally converting a compound of formula (I), wherein W and Z are both hydrogen, to a compound in which either or both of W and Z is a pharmaceutically acceptable cation, by reaction with a base or a salt containing the desired cations.

In process (a), derivatives of phosphoric acid having one to three hydroxy groups are replaced by halogen atoms, eg chlorine, such as phosphorus oxychloride (POCl 3 L are preferred for phosphorylation. Up to two of the hydroxy groups may also be substituted to form alkoxy groups optionally bearing additional substituents to Such phosphorus halide derivatives or phosphates are used under the usual neutral or alkaline conditions, the latter preferably requiring activation, for example with carbodiimide, eg dicyclohexylcarbodiimide, except when presented in the form of the anhydride.

When at least two of the hydroxyl groups in the phosphoric acid derivative are replaced by halogen, it is necessary, after reaction with the compound of formula (II), to remove the free halogens, by rather mild water hydrolysis, using, for example, 1 mol valent of water in a water-miscible solvent such as alcohol.

Substituted or unsubstituted alkoxy groups introduced with a phosphate can be hydrolyzed in a suitable aqueous medium in the presence of bases in a subsequent step. Aromatically substituted alkoxy groups, such as benzyloxy, may also be subjected to hydrogenolysis, preferably in the presence of a catalyst, according to the usual reductive cleavage techniques.

A preferred process for phosphorylating the intermediate compounds of the present invention comprises reacting a compound of formula (II), as defined above, with phosphorus oxychloride in the presence of a trialkyl phosphate and preferably at a temperature of about 0 ° C or lower.

Other useful methods for preparing the monophosphate involve reacting a compound of formula (II) with phosphorus oxychloride in dry pyridine.

The compound of formula (II) can be considered as an intermediate in the synthesis of the compounds of formula (I) and it can be prepared according to the procedures described in Swedish patent application 7509675-0.

Conversion of a compound of formula (III), by process (b), can be accomplished in several different ways, wherein the C1 group is converted to an amine group by ammonolysis. This method and the other methods well known in the art can be found in "Heterocyclic Compounds - Fused Pyrimidines Part II Purines edited by D.J. Brown (1971) published by Wiley - Interscience".

M may represent a halogen atom, mercapto or alkylthio group, which may be converted to a hydroxy group by hydrolytic methods described in the above book.

Compounds of formula (III) may be considered as intermediates in the synthesis of compounds of formula (I), and they may be prepared in an analogous manner according to process (a), which compounds in turn may be prepared analogously according to the procedures described in the Swedish patent application 7509675-O.

Pharmaceutically acceptable salts of 9- (2-hydroxyethoxymethyl) guanine monophosphate can be prepared by neutralizing the monophosphate in its acidic form with an equivalent (ie, equinormal) amount of a base such as a hydroxide, a bicarbonate, a carbonate containing the desired cation, i.e. sodium, potassium, ammonium, calcium, lithium, magnesium or aluminum. Alternatively, they may be prepared by the exchange reaction whereby a salt of the monophosphate is treated with a solution, preferably aqueous, of a salt containing the desired cation.

For example, the slightly soluble barium salt of the 9- (2-hydroxyethoxymethyl) guanine monophosphate is treated in aqueous suspension with sodium sulfate to remove the barium in the form of the highly insoluble barium sulfate, the sodium 9- (2-hydroxyethoxymethyl) guanine monophosphate remaining in solution.

The compounds prepared according to the invention may then be included in a pharmaceutical composition comprising a compound of formula (I), as defined above, together with a pharmaceutically acceptable carrier therefor.

Pharmaceutically acceptable carriers are materials which are useful for the purpose of administering the composition, as they may be solid, liquid or gaseous materials, which are otherwise inert and medically acceptable and are compatible with the active ingredient.

These pharmaceutical compositions may be administered orally, parenterally, used as a suppository or pessary, applied topically as an ointment, cream, aerosol or a powder, or given as eye or nose drops, depending on whether the preparation is used to treat viral infections. For internal infections, the compounds of formula (I) are administered at dose levels of 0.1 to 250 mg / kg, calculated as the free phosphate form, preferably 1.0 to 50 mg / kg mammalian body weight, and are used for humans in a unit dosage form. , for example, administered a few times daily as one or more unit doses, in an amount of 1 ~ 800 mg / kg per unit dose, preferably 1-250 mg / kg per unit dose, most preferably 100 ~ 200 mg per unit dose.

For parenteral administration, or topical administration as drops, for example for eye infections, compounds of formula (I) may be presented in aqueous solutions at a concentration of about 0.1-10% w / v, 0.1-7%, most preferably 0.2-5% w / v.

Alternatively, for infections of the eye or preferably other external tissue, such as the mouth and skin, topical preparations in the form of solutions, ointments or creams are preferred.

Concentrations of about 0.1 to 10%, preferably 0.3 to 6%, and most preferably 3% may be used.

The compounds prepared according to the present invention can be used in methods of treating viral infections in mammals, which method comprises administering an effective, non-toxic, antiviral amount of a compound of formula (I), as defined above.

As used herein, the term "effective non-toxic antiviral amount" refers to a predetermined amount of antiviral that is sufficient to be effective against the virus in vivo.

The invention is further illustrated by the following examples.

EXAMPLE 1 2: Phosphorus oxychloride (0.03 ml) was added in one portion to a stirred suspension of 2-chloro-9- (2-hydroxyethoxymethyl) -hypoxanthine (20 mg) in triethyl phosphate (0.3 ml).

The temperature was allowed to rise to 0 ° C for 30 minutes. The reaction mixture was then stirred at 0 ° C for 40 minutes and at + 5 ° C for 50 minutes. It was then poured onto ice and the pH was adjusted to 7 with 2N potassium hydroxide. The resulting solution was extracted twice with chloroform (2 x 2 ml). The pH of the aqueous phase was adjusted to 8-8.5 with 2N potassium hydroxide, and barium acetate (105 mg) was added. The resulting barium phosphate precipitate was removed by filtration. The supernatant was treated with a large excess of ethanol, precipitating crude barium 2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine monophosphate.

The solid was collected by filtration and suspended in ethanol. The ethanol-containing suspension was then heated on a steam bath for several minutes, cooled and filtered.

The collected precipitate was washed with anhydrous ether and dried to give 2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine monoiosphate (26 mg).

Ammonium sulfate (3.96 mg) was added to a stirred suspension of barium 2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine-7-monoiophate (7 mg) in water ( 0.5 ml). The landing was stirred at ambient temperature for 15 minutes, after which it was cooled in an ice bath. The barium sulfate precipitate was removed by filtration and washed with water (1 ml) and (10 ml). The combined filtrate and washings were evaporated under reduced pressure, and the resulting ethanolic residue was dissolved in methanol (3 ml). The methanol-lined stainless steel bomb, and the methanol (8 ml) saturated with the gaseous solution was transferred to a Teflon-shaped ammonia at ice bath temperature was also added to the bomb. The sealed bomb was placed in a 222 ° C oven for 4 hours, cooled and opened. The solvent was evaporated to a minimum volume. Stains of the remaining reaction mixture were applied to thin-layer plates of Eastman Chromatogram cellulose TLC, which were then developed in n-propanol water (70:30 v / v). The bands at Rf 0.16 and 0.34 were removed and suspended in Tris buffer (0.6 ml) at pH 8, and the cellulose was removed by filtration.

These bands were shown to contain 9- (2-hydroxy-ethoxymethyl) guanine monophosphate and 2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine monophosphate; by enzymatic dephosphorylation with alkaline phosphatase, 9- (2- hydroxyethoxymethyl) guanine or 2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine. Alkaline phosphatase (2 μl) from E. coli was added to the filtrate and the mixture was heated at 32 ° C for 2 hours. It was then examined by thin layer chromatography on Eastman Chromatogram cellulose plates in three solvent systems: (a) n-propanol: water (70: V / v) (b) water (c) n-propanol: concentrated ammonium hydroxide water (60: 30: 10 v / v) two spots were present in each system, corresponding to 9- (2-hydroxyethoxymethyl) guanine (A) and 2 -chloro-9- (2-hydroxyethoxymethyl) hypoxanthine (B). 7802140-9 10 15 20 25 30 35 8 Solvent system Rf (A) Rf (B) Rf for the reaction product (a) 0.51 0.64 0.51 and 0.65 (b) 0.68 0.67 , 67 and 0.97 (c) 0.51 0.71 0.51 and 0.71 EXAMPLE 2 2: (g: hv6r9§ieto§im§ethyl) guanine monophosphate Phosphorus oxychloride (0.76 ml) was added to a stirred, cooled (-10 ° C) mixture of 9- (2-hydroxyethoxymethyl) guanine (0.225 g) and triethyl phosphate (5 ml). The temperature of the reaction mixture was allowed to rise to 0 ° C for 30 minutes and kept at this temperature for 2 hours. It was then poured to reach a mixture of ice and water, and the pH was adjusted to 7 with 2N potassium hydroxide. The resulting solution was extracted twice with chloroform and once with ether.

The pH of the remaining aqueous solution was adjusted to 7.1 with 2N potassium hydroxide, after which it was lyophilized.

The resulting white solid was dissolved in water (7 ml), and methanol (7 ml) was added to precipitate the inorganic salts, which were then removed by filtration. Acetone (70 ml) was added to the filtrate, whereby a white gum precipitated. The gum was dissolved in water (7 ml), ethanol (7 ml) was added and the mixture was filtered.

A large excess of acetone (70 ml) was added, and again the rubber precipitated. The gum was dissolved in ethanol (about 20 ml) and the solvent was removed by equilibrium distillation to give a white powder (2.6 g), which was a mixture of inorganic salts and the desired phosphate. The solid was dissolved in water (10 ml) and transferred to a Bio-Gel P-2 column (74 Pm-37 Pm, 2.7 x 90 cm) and eluted with water. Most of the monophosphate was eluted in a 50 ml volume after 166 ml of the eluate had been collected, as shown by thin layer chromatography on Eastman Chromagramc) cellulose in n-propanol water (70: 30 volume / volume); Rf = 0.26 for 9- (2-hydroxyethoxymethyl) guanine phosphate and Rf '= 0.11 for potassium -9- (2-hydroxystoxymethyl) guanine phosphate. The eluate was lyophilized to give 0.28 g of a solid, which was shown by ultraviolet spectroscopy to contain 0.2 g of the monophosphate product.

EXAMPLE 3 In the case of β-β-β-β-β-α-β-α-β-α-β-α-β-α-β-α-β-α-β-α-β-α 6n hydrochloric acid. The product was adsorbed on 14 ml of cat charcoal (Fischer 5-69OB, 297 Fm - 74 μm, acid washed Pa: deactivated with toluene). The carbon was washed well with the lotion and eluted with 70 ml of 50% aqueous ethane® 1I containing 2% concentrated ammonium hydroxide. The solvent was evaporated under reduced pressure to give ammonium 9- (2-hydroxyethoxymethyl) guanine monophosphate (0.43 g); Rf = 0.30 on Eastman Cellulose in n-propanol: water (70: 30 v / v).

EXAMPLE 4 Disatrial: Phosphorus oxychloride (54 ml) was added over 3 hours to a Guif ear, cooled (330 ° to -20 ° C) mixture of e- (zmydroxy-ethoxymethyl) guanine ) and triethyl phosphate (250 ml).

The temperature of the reaction mixture was allowed to rise to 0 ° C for 45 minutes and kept at this temperature for a further 45 minutes. It was then poured into a mixture of ice and water and its pH was adjusted to about 1 with 2N sodium hydroxide. The resulting solution was extracted once with chloroform and once with ether. The pH of the remaining aqueous solution was adjusted to 6.8 with 2N sodium hydroxide and then to 7.3 with 10N sodium hydroxide, whereby a final volume of 2.5 liters was obtained.

The neutralized solution was transferred to a column containing 2000 g of Dowex 1 x 8, which had been equilibrated with 50 mM KHCO 3. The elution was performed with a 30 liter linear gradient of 50-500 mM KHCO3, followed by a 30 liter wash with 500 mM KHCO3. The fractions containing the product were combined, and most of the KHCO 3 was removed by adding Dowex 50-H + and removing CO 2 under vacuum. The volume was reduced to 2 liters in vacuo and the product precipitated at 4 ° C by adding 10 liters of acetone. The dried precipitate, 55 g, was transferred to a 10 x 110 cm Bio-Gel P-2 column and eluted with water, 22 g of solid was obtained. The material was recrystallized at 400 as the hydrogen salt from a pH 3 solution of water, and more material was obtained from the mother liquor by crystallization from 20% ethanol at pH 3. The hydrogen salt was dissolved in a minimal volume of water, the pH of which was brought to 8.5 with NaOH, and precipitated with 2 volumes of ethanol at 4 ° C. This precipitate was dissolved in 100 ml of water and precipitated with 9 volumes of ethanol at 400 to give 15.1 g of 9- (2-hydroxyethoxymethyl) guanine monophosphate disodium salt dihydrate.

Purity was confirmed by elemental analysis, high pressure liquid chromatography and UV spectra.

Empirical formula: C 8 H 12 N 5 O 5 P 2 Na 2 H 2 O Calculated: 24.94% C 3.66% H 18/19% N 8.04% P Found: 25.20% C 3.63% H 18.10% N 7.89% P UV spectra: Solvent F max EA min sh 0.1 M HCl 254 12970 225 272 pH 7 249 14060 219 266 0, lm NaOH 255-264 11760 228 High pressure liquid chromatography unit = 99% Base / phosphate ratio = 1.00 / 1.01

Claims (8)

10 15 20 25 30 7802140-9 ll PATENT REQUIREMENTS A process for the preparation of a 9-hydroxyethoxymethylguanine derivative of the formula (I) N / I (I) N ÅH2. the same or different and each represents a hydrogen atom or pharmaceutically acceptable cation, characterized in that (a) a compound of formula (II) HN / | / “> \ NN (In H2 N H2.O.CHz.CH2.OH reacting with a phosphorylating agent to give a compound of formula (I), wherein W and Z are hydrogen atoms, or (b) a compound of formula (III) OH NI /, (/// Ammonolysis to give a compound of formula (I) is subjected to ammonolysis to give a compound of formula (I). and optionally converting a compound of formula (I), wherein W and Z are both hydrogen, to a compound in which either or both of W and Z is a pharmaceutically acceptable cation, by reaction with a base or a salt containing the required cation. Process according to Claim 1 (a), characterized in that the phosphorylating agent is a derivative of phosphoric acid. 3. A process according to claim 2, characterized in that the derivative of phosphoric acid has 1 to 3 hydroxyl groups replaced by halogen atoms. 4. A process according to claim 3, characterized in that the phosphoric acid derivative is phosphorus oxychloride. 5. A process according to claim 4, characterized in that the compound of formula (II) is reacted with phosphorus oxychloride in the presence of a trialkyl phosphate at a temperature of 0 ° C or lower. 6. A process according to claim 4, characterized in that the compound of formula (II) is reacted with phosphorus oxychloride in dry pyridine. 7. A process according to any one of claims 1-6, characterized in that the base with which a compound of formula (I), wherein W and Z are both hydrogen, is reacted is a hydroxide, a bicarbonate or a carbonate containing the required pharmaceutically acceptable cation. 8. A process according to claim 7, characterized in that the cation required is sodium, potassium, ammonium, calcium, lithium, magnesium or aluminum.